The technique of density gradient centrifugation is widely used to isolate cellular macromolecules and organelles. Various kinds of devices have been designed to facilitate collection of fractions from density gradients in centrifuge tubes. These devices are employed in methods that generally involve displacement of the density gradient up or down the centrifuge tube by either a puncturing or a nonpuncturing procedure.
The earliest and simplest puncturing method was to pierce the bottom of the centrifuge tube with a small-bore hollow needle and to collect the emerging drops. Such a method is described in Weigle, J., Meselson, M., and Pagien, K. (1959) J. Mol. Biol. 1, 379-386. Improvements on this basis method involve (i) controlling the flow rate of the effluent or the speed at which air, distilled water, or mineral oil enter at the top, (ii) preventing the entry of air bubbles into the gradient, and (iii) reducing turbulent flow at the abrupt and narrow juncture between the tube bottom and the bore of the needle. These improvements are described in Szybalski, W. (1960) Experientia 16, 164; Heckly, R. J. (1960) Anal. Biochem. 1, 97-102; Englund, P. T., Smith, H. O., and Sandbeck, E. (1971) Anal. Biochem. 40, 490-493; Noll, H. (1969) Anal. Biochem. 27, 130-149; Tan, K. B. (1972) Anal. Biochem. 45, 306-308; Hopkins, T. R. (1973) Anal. Biochem. 53, 339-341; Clark, R. W., Carnes, J. D., and Arrighi, F. E. (1975) Anal. Biochem. 67, 139-146. Another puncturing method makes use of a small-diameter cannula which is inserted through the side or bottom of a centrifuge tube. A dense solution is pumped into the cannula displacing the gradient upward, thus delivering fractions from the top. Reference is made to the following articles for a further description of this technique: Hopkins, T. R. (1973) Anal. Biochem. 53, 339-341; Clark, R. W., Carnes, J. D., and Arrighi, F. E. (1975) Anal. Biochem. 67, 139-146; Brakke, M. K. (1963) Anal. Biochem. 5, 271-283; Bubel, H. C., and Riley, B. P. (1968) Anal. Biochem. 22, 335-337; Bresch, H., and Meyer, H. (1973) Anal. Biochem. 53, 199-207; and Romani, R. J., and Fisher, L. K. (1967) Anal. Biochem. 21, 333-335.
Nonpuncturing procedures usually employ a long narrow capillary tube that is carefully lowered down through the middle of a gradient to the bottom of the centrifuge tube. The capillary tube serves as an inlet to pump in air or to introduce a dense solution, thereby forcing the gradient upward and directing the fractions through a top outflow orifice. (See Oumi, T., and Osawa, S. (1966) Anal. Biochem. 15, 539-541; Liedtke, R., and Mosebach, K. O. (1974) Anal. Biochem. 62, 377-385 as well as the Hopkins article referred to above.) Suction may also be applied to the capillary tube to withdraw the gradient from the bottom of the centrifuge tube. This technique is described further in Fox, T. O., and Pardee, A. B. (1970) Science 167, 80-82. Another procedure uniquely eliminates the need to insert a capillary tube by utilizing a narrow recovery/delivery channel that has been drilled the length of, and thus is already incorporated into, the thick wall of a specially fabricated centrifuge tube. Reference is made to Leif, R. C. (1968) Anal. Biochem. 25, 271-282. Fractionation by piston displacement involves forcing a specially tipped piston into a centrifuge tube from above and, with the use of continuously applied pressure, the tube contents are gradually displaced through the tip. Fractions are conveyed by collection tubing that is threaded through a channel in the piston shaft. (See Coombs, D. H. (1975) Anal. Biochem. 68, 95-101.) Another collecting apparatus consists of a plastic disk which is suspended through a hole in its center onto the flared end of a stretch of coiled tubing. The plastic disk is carefully pressed to the surface of the gradient until it becomes attached by surface tension. Suction is applied to the free end of the coiled tubing and, as the fractions are collected, the coiled tubing stretches while the descending fluid surface pulls the plastic disk with it. This technique is described in Germain, G. S. (1974) Anal. Biochem. 57, 89-92.
Although they are proposed and designed to improve the efficiency and ease of sample removal from density gradients, these methods and devices suffer from certain disadvantages. By retrieving gradients with puncturing techniques, mixing of contents can occur at the bottom of the tube owing to nonideal turbulent flow and contamination of successive fractions can arise when densely banded materials are collected through the slightly protruding narrow-bore needle. Movement of the entire gradient contents by displacement, either by puncturing or by nonpuncturing techniques, can introduce artifacts by wiping off material which has adhered to the tube wall and by dislodging material which has sedimented to the tube bottom. Expense becomes a consideration as tubes are destroyed by puncturing and as specialized accessory equipment is employed to ensure high performance and circumvent some of the previously mentioned problems. Many of the collecting devices are themselves highly complex and difficult to fabricate. Although inexpensive and simple to construct, the very design of the upper surface-following device described in the Germain article referred to above is a serious shortcoming. Severe mixing can occur because the surface fluid, before being drawn up into the bore of the collection tubing, is required to traverse radially across the flat disk to the central collecting point and move downward beneath the slightly protruding flared end of the tubing.
Patent references of possible interest include U.S. Pat. Nos. 275,134 (Burton); 536,858 (Donato); 1,591,923 (Lebherz); 3,955,423 (Ohringer); 3,960,727 (Hochstrasser); 3,972,683 (Lake); and 4,197,735 (Munzer et al), although, in general, these references are not concerned with fractionation of liquids.